Modulation of the Major Paths of Carbon in Photorespiratory Mutants of Synechocystis

Abstract

Recent studies using transcript and metabolite profiles of wild-type and gene deletion mutants revealed that photorespiratory pathways are essential for the growth of Synechocystis sp. PCC 6803 under atmospheric conditions. Pool size changes of primary metabolites, such as glycine and glycolate, indicated a link to photorespiration. The ¹³C labelling kinetics of primary metabolites were analysed in photoautotrophically grown cultures of Synechocystis sp. PCC 6803 by gas chromatography-mass spectrometry (GC-MS) to demonstrate the link with photorespiration. Cells pre-acclimated to high CO₂ (5%, HC) or limited CO₂ (0.035%, LC) conditions were pulse-labelled under very high (2% w/w) ¹³C-NaHCO₃ (VHC) conditions followed by treatment with ambient ¹²C at HC and LC conditions, respectively. The ¹³C enrichment, relative changes in pool size, and ¹³C flux of selected metabolites were evaluated. We demonstrate two major paths of CO₂ assimilation via Rubisco in Synechocystis, i.e., from 3PGA via PEP to aspartate, malate and citrate or, to a lesser extent, from 3PGA via glucose-6-phosphate to sucrose. The results reveal evidence of carbon channelling from 3PGA to the PEP pool. Furthermore, ¹³C labelling of glycolate was observed under conditions thought to suppress photorespiration. Using the glycolate-accumulating ΔglcD1 mutant, we demonstrate enhanced ¹³C partitioning into the glycolate pool under conditions favouring photorespiration and enhanced ¹³C partitioning into the glycine pool of the glycine-accumulating ΔgcvT mutant. Under LC conditions, the photorespiratory mutants ΔglcD1 and ΔgcvT showed enhanced activity of the additional carbon-fixing PEP carboxylase pathway. With our approach of non-steady-state ¹³C labelling and analysis of metabolite pool sizes with respective ¹³C enrichments, we identify the use and modulation of major pathways of carbon assimilation in Synechocystis in the presence of high and low inorganic carbon supplies.